title: Highly divergent strains of porcine reproductive and respiratory syndrome virus incorporate multiple isoforms of nonstructural protein 2 into virions.
abstract: Viral structural proteins form the critical intermediary between viral infection cycles within and between hosts, function to initiate entry, participate in immediate early viral replication steps, and are major targets for the host adaptive immune response. We report the identification of nonstructural protein 2 (nsp2) as a novel structural component of the porcine reproductive and respiratory syndrome virus (PRRSV) particle. A set of custom α-nsp2 antibodies targeting conserved epitopes within four distinct regions of nsp2 (the PLP2 protease domain [OTU], the hypervariable domain [HV], the putative transmembrane domain [TM], and the C-terminal region [C]) were obtained commercially and validated in PRRSV-infected cells. Highly purified cell-free virions of several PRRSV strains were isolated through multiple rounds of differential density gradient centrifugation and analyzed by immunoelectron microscopy (IEM) and Western blot assays using the α-nsp2 antibodies. Purified viral preparations were found to contain pleomorphic, predominantly spherical virions of uniform size (57.9 nm ± 8.1 nm diameter; n = 50), consistent with the expected size of PRRSV particles. Analysis by IEM indicated the presence of nsp2 associated with the viral particle of diverse strains of PRRSV. Western blot analysis confirmed the presence of nsp2 in purified viral samples and revealed that multiple nsp2 isoforms were associated with the virion. Finally, a recombinant PRRSV genome containing a myc-tagged nsp2 was used to generate purified virus, and these particles were also shown to harbor myc-tagged nsp2 isoforms. Together, these data identify nsp2 as a virion-associated structural PRRSV protein and reveal that nsp2 exists in or on viral particles as multiple isoforms.
authors.affiliation: Virus and Prion Research Unit, USDA-ARS-National Animal Disease Center, Ames, Iowa, USA.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Kappes
authors.forename: Cathy L
authors.initials: CL
authors.lastname: Miller
authors.forename: Kay S
authors.initials: KS
authors.lastname: Faaberg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019143:Evolution, Molecular; D008969:Molecular Sequence Data; D019318:Porcine Reproductive and Respiratory Syndrome / Q000821:virology*; D019316:Porcine respiratory and reproductive syndrome virus / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013552:Swine; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*
chemical_list: D020033:Protein Isoforms; D017361:Viral Nonstructural Proteins